Abstract

Trophic interactions between pulmonary epithelial and mesenchymal cell types, known as the epithelial-mesenchymal trophic unit (EMTU), are crucial in lung development and lung disease. Transforming growth factor (TGF)-beta is a key factor in mediating these interactions, but it is expressed in a latent form that requires activation to be functional. Using intact fetal tracheal tissue and primary cultures of fetal tracheal epithelial cells and fibroblasts, we demonstrate that a subset of integrins, alpha(v)beta(6) and alpha(v)beta(8), are responsible for almost all of the TGF-beta activation in the EMTU. Both alpha(v)beta(8) and alpha(v)beta(6) contribute to fetal tracheal epithelial activation of TGF-beta, whereas only alpha(v)beta(8) contributes to fetal tracheal fibroblast activation of TGF-beta. Interestingly, fetal tracheal epithelial alpha(v)beta(8)-mediated TGF-beta activation can be enhanced by phorbol esters, likely because of the increased activity of MT1-MMP, an essential co-factor in alpha(v)beta(8)-mediated activation of TGF-beta. Autocrine alpha(v)beta(8)-mediated TGF-beta activation by fetal tracheal fibroblasts results in suppression of both transcription and secretion of hepatocyte growth factor, which is sufficient to affect phosphorylation of the airway epithelial hepatocyte growth factor receptor, c-Met, as well as airway epithelial proliferation in a co-culture model of the EMTU. These findings elucidate the function and complex regulation of integrin-mediated activation of TGF-beta within the EMTU.

The integrin αvβ8 is expressed by fetal tracheal epithelial cells and fibroblasts. A: RT-PCR for the β8 integrin subunit (top) and β-actin (bottom) was performed from total RNA harvested from fetal tracheal fibroblasts (lane 1), fetal tracheal epithelial cells (lane 2), wild-type SW480 cells stably transfected with a β8 expression construct (lane 3), SW480 cells (lane 4), and a control with no added cDNA (lane 5). Shown are bands migrating at the appropriate sizes for the amplification products. Flow cytometry of passage 1 fetal tracheal epithelial cells (B) and fetal tracheal fibroblasts (C) was performed using either no primary antibody (black bars), anti-β6 (horizontal hatched bars), or anti-β8 (vertical hatched bars). Results are expressed as the mean fluorescence intensity in arbitrary fluorescence units (log10). Immunoprecipitation of biotin surface-labeled fetal tracheal (FT) epithelial cells (D) or fetal tracheal fibroblasts (E) using no primary antibody or anti-integrin subunit and complex specific antibodies against αv (L230), β1 (P5D2), β3 (AP3), αvβ5 (P1F6), αvβ6 (E7P6), or αvβ8 (14E5). Antibodies used are indicated below each lane. The migration of the molecular size markers is indicated on the right, and the subunits corresponding to each band are indicated on the left. The experiments shown are representative of at least three independent experiments giving similar results.

Autocrine αvβ8-mediated activation of TGF-β activation contributes to the regulation of the myofibroblast phenotype. A: Western blot for α-SMA of fetal tracheal fibroblast cell lysates that had been treated for 24 hours with no treatment (lane 1), anti-pan TGF-β (lane 2), anti-β8 (lane 3), or recombinant active TGF-β1 (lane 4). A lysate of fetal tracheal epithelial cells is added as a negative control (lane 5). Shown at the left is a representative experiment and at the right is densitometry performed from three independent experiments. Filled bar is no treatment, open bar is anti-TGF-β, horizontal crosshatched bar is anti-β8, and vertical hatched bar is recombinant active TGF-β. *P < 0.05. B: Collagen gel contraction assay of fetal tracheal fibroblasts treated with no treatment (1), anti-TGF-β (2), anti-β8 (3), or recombinant active TGF-β (4). On the left photomicrographs depict the well containing the collagen gels at 0 (top) and 72 hours (bottom). On the right is the average maximum gel diameter taken from three independent experiments shown as percent increase in size compared to the no treatment control. Filled bar is no treatment, open bar is anti-TGF-β, horizontal crosshatched bar is anti-β8, and vertical hatched bar is recombinant active TGF-β. *P < 0.05, **P < 0.001. Shown is SE.